The broad objectives of this project are to define the requirements for the development and maintenance of regulatory T cells (Treg), the mechanisms of action of Treg, and the potential of these cells as therapy in a mouse model of type 1 diabetes. There are several unique aspects of this project, i) experimental systems that rely on antigen-specific Treg expressing an easily identifiable T cell receptor (TCR); ii) an emphasis on studying the biology and mechanisms of action of Treg in vivo; and iii) the use of methods for analyzing gene expression and for manipulating genes in primary lymphocytes to define the requirements and mechanisms of action of Treg. The specific aims of the project are the following: 1. Development of regulatory T cells : We will define the phenotypes, functional capabilities, and cytokine dependence of Treg expressing the DO11.10 TCR in TCR transgenic mice crossed with mice expressing a cell-associated form of the cognate antigen, OVA, in tissues (islet beta cells) or a secreted form in the serum. This aim will establish if developmental exposure to different forms of an antigen generates similar or distinct Treg populations. 2. Role of costimulation and antigen in the generation and maintenance of Treg: Using B7 antagonists and B7-knockout mice, we will establish the role of B7 molecules in the thymic development, peripheral survival, and cycling of OVA-specific Treg. We will also define the importance of antigen by transferring Treg into recipients that do and do not express different forms of the antigen. We will use gene expression profiling to identify molecules that may be responsible for the effects of costimulators or antigen on Treg, and express candidate genes, identified from expression analyses, in Treg to demonstrate their role in responses to B7 or antigen. 3. Influence of Treg on the development of diabetes: Using a transgenic mouse model of diabetes, we will define the ability of transferred Treg to prevent or treat disease, and when in the course of disease these cells are efficacious. We will also develop protocols for generating large numbers of antigen-specific Treg for therapeutic trials. 4. Mechanism of action of Treg: We will define the changes induced in pathogenic responder cells by exposure to antigen-specific Treg in vivo and in vitro, as one approach to determining how Treg work. We will also identify genes induced in Treg by activation, under conditions that induce maximal suppressive activity, and examine the roles of these genes in Treg function. Together, the proposed studies will define the basic biology of regulatory T cells, their potential value for therapy, and ways to maximize their survival and function in vivo. These animal studies may provide the pre-clinical foundation for the optimal use of Treg in immunotherapy.